Identification, characterization, and structure of a tRNA splicing enzyme RNA 5′-OH kinase from the pathogenic fungi Mucorales

GTP is the preferred phosphate donor for MciKIN. Reaction mixtures (10 µL) containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM DTT, 1 mM MgCl₂, 100 nM (1 pmol) 10-mer 5′-OH RNA substrate 3′-labeled with Cy5, 5 nM (50 fmol) MciKIN, and 0, 1, 10, 100, or 1000 µM of the indicated NTP were incubated at 37˚C for 5 min. MciKIN was omitted from the reaction mixture in lane –E. The reactions were quenched with formamide/EDTA, and the products were analyzed by urea-PAGE. The positions of the 10-mer _HORNA substrate and the 10-mer pRNA product are indicated.